The efficiency with which the immune system cures or protects individuals from infectious disease has always been intriguing to scientists, as it has been believed that it might be possible to activate the immune system to combat other types of diseases. Such diseases include cancer, AIDS, hepatitis and infectious disease in immunosuppressed patients. While various procedures involving the use of antibodies have been applied in those types of diseases, few if any successful attempts using cytotoxic T lymphocytes have been recorded. Theoretically, cytotoxic T lymphocytes would be the preferable means of treating the types of disease noted above. However, no useful in vivo procedures have been available to specifically activate cytotoxic T lymphocytes.
Cytotoxic T lymphocytes (CTLs), which are also called cytotoxic T cells or CD8 cells, represent the main line of defense against viral infections. CTLs specifically recognize and kill cells which are infected by a virus. Thus, the cost of eliminating a viral infection is the accompanying loss of the infected cells. The T cell receptors on the surface of CTLs cannot recognize foreign antigens directly. In contrast to antibodies, antigen must first be presented to the receptors.
The presentation of antigen to T cells is accomplished by major histocompatibility complex (MHC) molecules of the Class I type. The major histocompatibility complex (MHC) refers to a large genetic locus encoding an extensive family of glycoproteins which play an important role in the immune response. The MHC genes, which are also referred to as the HLA (human leucocyte antigen) complex, are located on chromosome 6 in humans. The molecules encoded by MHC genes are present on cell surfaces and are largely responsible for recognition of tissue transplants as “non-self”. Thus, membrane-bound MHC molecules are intimately involved in recognition of antigens by T cells.
MHC products are grouped into three major classes, referred to as I, II, and III. T cells that serve mainly as helper cells express CD4 and are primarily restricted by Class II molecules, whereas CTL-(CD8−) expressing cells, which mostly represent cytotoxic effector cells, interact with Class I molecules.
Class I molecules are membrane glycoproteins with the ability to bind peptides derived primarily from intracellular degradation of endogenous proteins. Complexes of MHC molecules with peptides derived from viral, bacterial and other foreign proteins comprise the ligand that triggers the antigen responsiveness of T cells. In contrast, complexes of MHC molecules with peptides derived from normal cellular products play a role in “teaching” the T cells to tolerate self peptides, in the thymus. Class I molecules do not present entire, intact antigens; rather, they present peptide fragments thereof, “loaded” onto their “peptide binding groove”.
For many years, immunologists have hoped to raise specific cytotoxic cells targeting viruses, retroviruses and cancer cells. While targeting against viral diseases in general may be accomplished in vivo by vaccination with live or attenuated vaccines, no similar success has been achieved with retroviruses or with cancer cells. Moreover, the vaccine approach has not had the desired efficacy in immunosuppressed patients. One way around this difficulty would be to immunize a healthy individual, isolate the CTLs from this individual, and inject these CTLs into the disease-afflicted person.
However, this experimental protocol is not always useful, as it is neither practical (nor ethical) in many circumstances to endeavor to immunize healthy individuals with tumor cells. Furthermore, it is problematic, at best, to endeavor to activate CTLs to recognize abnormal cells expressing abnormally high levels of peptides that are expressed on normal cells in lower quantities in normal, healthy individuals.
The use of mouse strains (including transgenic strains) to generate activated CTLs has not always been practical, particularly if the murine strain is unable to raise an immunologic response to the immunogen. Failure to raise an immunologic response may be due either to failure of the murine immune system to recognize the antigen, or its failure to generate activated cells that are compatible with the intended recipient of activated CTLs for therapeutic purposes.
For example, it has been observed that peptides are unique for a given MHC; in other words, certain antigenic peptides bind preferentially to particular MHC species and do not bind well to others, even in the absence of the “preferred” MHC molecule. Furthermore, MHC molecules are highly polymorphic, which fact generates at least two problems. First, the CTLs of an individual can only interact with peptides bound to precisely those three to six Class I molecules present in that individual. Second, CTLs react violently with all Class I molecules which are different from those expressed in the individual from whom the CTLs are obtained, regardless of what peptides the Class I molecules contain. This reactivity has been observed for some time and is termed allo-reactivity. It is the underlying cause of the immune rejection of transplanted organs.
Thus, apart from the rather heroic experimental protocol in which one individual is used as the donor of activated CTLs to another individual, it is difficult to find two unrelated persons with the exact same setup of Class I molecules. For this reason, at least one researcher has taken the rather non-specific approach of “boosting” existing CTLs by incubating them in vitro with IL-2, a growth factor for T cells. However, this protocol (known as LAK cell therapy) will only allow the expansion of those CTLs which are already activated. As the immune system is always active for one reason or another, most of the IL-2 stimulated cells will be irrelevant for the purpose of combating the disease. In fact, it has not been documented that this type of therapy activates any cells with the desired specificity. Thus, the benefits of LAK cell therapy are controversial at best, and the side effects are typically so severe that many studies have been discontinued.
Class I molecules bind peptides in a specific manner. All peptides have to be about 8-11 amino acids in length and their sequences must fit the peptide-binding pocket of the Class I molecules. In this respect, Class I molecules display some resemblance to antibodies. However, while a given antibody tends to bind only one antigen, a given Class I molecule can bind many hundred different peptides. As the number of viruses and other pathogens is quite large, it is apparent that our immune defense would be poor if we had only a single Class I molecule, even if it is capable of binding and altering many different peptides. For this reason, all humans have between three and six different Class I molecules, which can each bind many different types of peptides. Accordingly, the CTLs can recognize many thousands of peptides bound to one or another Class I molecule.
As selection seems to be the dominant force in evolution, pathogens emerge which cannot be recognized efficiently by the immune system. Thus, for example, a viral sequence, which gives rise to peptides that bind efficiently to a variety of Class I molecules, may mutate such that it is not recognized by any of the three to six Class I molecules present in an individual. This virus may therefore not be recognized by the immune system and may consequently cause the death of the affected individual. If all individuals had an identical set of Class I molecules, such a virus might conceivably eliminate an entire species.
However, individual variation is a safeguard against that possibility, as some 100 different forms of Class I molecules are present in the population.
If Class I molecules can bind a variety of peptides, including peptides derived from our own cellular proteins, one may wonder why the CTLs of the immune system do not recognize and destroy our own tissues. While the answer to this question is not entirely clear, two distinct mechanisms are presently believed to be operating. First, CTLs that can react with self peptides are eliminated in the thymus. Second, CTLs become non-responsive (anergic) to self peptides in the peripheral organs of the immune system. Since every possible type or epitope of cellular proteins is not synthesized by the cells in the thymus, the second mechanism would appear to be the more likely explanation. This mechanism appears to be operational for the level of self peptides normally encountered. If this level is increased by some means, it can be shown that individuals do indeed have CTLs that can recognize and destroy cells expressing self peptides. This latter observation is significant with regard to the concept of using the immune system to eliminate tumor cells.
Recently, it has become apparent that mutant and wild-type peptides derived from cellular oncogene proteins can be recognized by CTLs. This suggests that self peptides encoded by non-mutant genes, in addition to the peptides encoded by mutant genes, can be potential targets for T cell responses against tumor cells. (See, e.g., Melief and Kast, Curr. Op. Immunol. 5: 709-713 (1993); Boon, Adv. Cancer Res. 58: 177-210 (1992); Van der Bruggen, et al., Curr. Op. Immunol. 4: 608-612 (1992).)
Irrespective of the mode of activity, it is evident that the CTL response with respect to various tumor antigens is deficient in many cases. It would be desirable to stimulate the immune response in these individuals to respond to appropriate tumor antigens and thereby eliminate the cells and tissues so affected. Further, as there is no currently available vaccine for malignancies such as breast cancers, it is desirable to establish such a vaccine, preferably based on a range of antigenic determinants.
Accordingly, it is an object of the present invention to provide agents that strengthen or boost the ability of the cellular immune system to fight tumors and other malignancies. It is a further object to provide pharmaceutical compositions that strengthen or boost the cellular immune system for fighting tumor-related disease processes, both with reference to therapeutic and prophylactic uses.
These and other objects and advantages of the present invention, as well as additional inventive features, will be apparent from the description of the invention provided herein.